Dissociated cell cultures of dopaminergic neurons from embryonic rat mesencephalon were grown in completely defined serum-free medium. When exposed to increased levels of extracellular potassium, these cultures are damaged mainly via NMDA-receptors. Reasoning that cells in vivo could be exposed to an excessive concentration of extracellular K+ during ischemia or anoxia, we investigated the sequence of events involved in neurotoxicity following exposure to K+ and compared these to the events following exposure to NMDA. Damage following exposure to K+ is mostly blocked by D,L-APV (200fM), indicating a role for the NMDA receptor. Damage significantly decreases when cells are exposed to increased potassium in Ca++-free buffer and does not occur in Ca++-free buffer in the present of inhibitor of release of Ca++ from intracellular stores (dantrolene), indicating for role of Ca++ in this process. Inhibitors of cyclooxygenase (indomethacin), lipoxygenase (nordihydroguaiaretic acid), phospholipase A2 (mepacrine), nitric oxide synthase (nitro-L- arginine) were without effect on K+ induced culture damaged. Inhibitor of protein kinase C (calphostin C) has very little effect. However, a calmodulin antagonist (W-5; 150microM) significantly protected the culture and an inhibitor of Ca++ calmodulin-dependent protein kinase (KN- 62) has also significant but less effect. In the same time, neither W-5 nor KN-62 had any effect on damage induced by NMDA. Analysis of aminoacid pool of extracellular medium after 20 minutes of exposure of culture to high K+ shows that concentration of glutamate in the medium reaches toxic level (5microM). In the same time it is only 0.9microM in the present of 10fM KN-62 and 0.7microM in the present of 150microM W-5. No one from the inhibitors of mentioned above enzymes decrease significantly concentration of glutamate in extracellular medium.